Characterization and application of newly developed polymorphic microsatellite markers in the Ezo red fox (Vulpes vulpes schrencki).

The Ezo red fox (Vulpes vulpes schrencki), a subspecies endemic to Hokkaido island, Japan, is a known host species for the tapeworm Echinococcus multilocularis. To develop tools for molecular ecological studies, we isolated 28 microsatellite regions from the genome of Ezo red fox, and developed 18 polymorphic microsatellite markers. These markers were characterized using 7 individuals and 22 fecal samples of the Ezo red fox. The number of alleles for these markers ranged from 1 to 7, and the observed heterozygosity, estimated on the basis of the genotypes of 7 individuals, ranged from 0.29 to 1.00. All markers, except DvNok5, were in Hardy-Weinberg equilibrium (P > 0.05), and no linkage disequilibrium was detected among these loci, except between DvNok14 and DvNok28 (P = 0.01). Moreover, six microsatellite loci were successfully genotyped using feces-derived DNA from the Ezo red fox. The markers developed in our study might serve as a useful tool for molecular ecological studies of the Ezo red fox.

Development of microsatellite markers for Cinnamomum camphora (Lauraceae).

PREMISE OF THE STUDY: Cinnamomum camphora is an evergreen tree distributed in southern Japan, Taiwan, and southeastern China. Because of its vast utilization and cultivation, the natural distribution area of this species has been controversial. METHODS AND RESULTS: I isolated and characterized 22 microsatellite loci in C. camphora. Levels of polymorphism were evaluated in 104 adult trees from three populations in Japan: Meiji Jingu (Shinto Shrine), Kajiya Plantation, and Manazuru Peninsula. The mean number of alleles per locus ranged from 4.1 to 8.0 among populations. The mean observed and expected heterozygosities per population ranged from 0.53 to 0.60 and 0.55 to 0.68, respectively. CONCLUSIONS: All of 22 loci showed a clear and strong single band for each allele, and revealed a useful degree of polymorphism. The microsatellite markers described here will be useful to study the history, population dynamics, mating system, and genetic structure of C. camphora.

A hybrid zone dominated by fertile F1s of two alpine shrub species, Phyllodoce caerulea and Phyllodoce aleutica, along a snowmelt gradient.

In alpine ecosystems, the steep environmental gradients produced by the difference in snowmelt timing create a dynamic selective regime for alpine plants. As these gradients directly alter flowering phenology, they can affect pollen-mediated gene flow among populations of single and related species. In northern Japan, we found a hybrid zone dominated by fertile F(1)s of two alpine shrub species, Phyllodoce caerulea and P. aleutica, along a snowmelt gradient. Seed germination confirmed the fertility of F(1) hybrid, making the rarity and absence of backcross and F(2) plants puzzling. The long-term clonal perpetuation of F(1) hybrids (at least a few thousand years ago) contributes the maintenance of this unique hybrid zone. The distribution patterns of chloroplast DNA haplotypes suggest that F(1) formation might be caused by directional pollen flow between parental species along the snowmelt gradient. Based on these results, we discuss the ecological and evolutionary significance of this unique hybrid zone.

Heating applicator based on reentrant cavity with optimized local heating characteristics.

PURPOSE: A reentrant-cavity-based applicator can produce a concentrated electric field between reentrant electrodes for localized heating. However, this field is inadequate for treating early small tumors localized in the head and neck. In order to safely heat such well-localized lesions, the electric field distribution should be more localized. MATERIALS AND METHODS: In order to achieve localized heating, four parameters of the reentrant cavity (applicator height, outer diameter, reentrant diameter, and reentrant gap size), which influence the distribution of the electric field produced in the reentrant gap, are optimized using the Taguchi method. The variation in the heating characteristics affected by the size of the heating object is estimated using the signal-to-noise ratio (SNR) index. In this study, the electromagnetic field distributions in a cylindrical phantom and an oblate sphere phantom are analyzed by the three-dimensional finite element method, and the full width at half height (FWHH) of the specific absorption rate (SAR) distribution in the reentrant gap is evaluated. RESULTS: It is shown that the optimized applicator yields both the maximum SNR and minimum mean FWHH, and the sizes of the heating region in the phantom expressed using the averaged FWHH values of the SAR distribution are 60 and 80 mm along the radial and long-axis directions of the applicator, respectively. CONCLUSIONS: A heating region can be robustly and optimally localized by using the Taguchi method and considering the variation in the size of the heating object.

Male-biased hermaphrodites in a gynodioecious shrub, Daphne jezoensis.

Gynodioecy, a state where female and hermaphrodite plants coexist in populations, has been widely proposed an intermediate stage in the evolutionary pathway from hermaphroditism to dioecy. In the gynodioecy-dioecy pathway, hermaphrodites may gain most of their fitness through male function once females invade populations. To test this prediction, comprehensive studies on sex ratio variation across populations and reproductive characteristics of hermaphrodite and female phenotypes are necessary. This study examined the variation in sex ratio, sex expression, flower and fruit production and sexual dimorphism of morphological traits in a gynodioecious shrub, Daphne jezoensis, over multiple populations and years. Population sex ratio (hermaphrodite:female) was close to 1:1 or slightly hermaphrodite-biased. Sex type of individual plants was largely fixed, but 15% of plants changed their sex during a 6-year census. Hermaphrodite plants produced larger flowers and invested 2.5 times more resources in flower production than female plants, but they exhibited remarkably low fruit set (proportion of flowers setting fruits). Female plants produced six times more fruits than hermaphrodite plants. Low fruiting ability of hermaphrodite plants was retained even when hand-pollination was performed. Fruit production of female plants was restricted by pollen limitation under natural conditions, irrespective of high potential fecundity, and this minimised the difference in resources allocated to reproduction between the sexes. Negative effects of previous flower and fruit production on current reproduction were not apparent in both sexes. This study suggests that gynodioecy in this species is functionally close to a dioecious mating system: smaller flower production with larger fruiting ability in female plants, and larger flower production with little fruiting ability in hermaphrodite plants.

Mechanism for adaptive modification during cold acclimation of phospholipid acyl chain composition in Tetrahymena. I. Principal involvement of deacylation-reacylation.

The purpose of the present studies with in vivo isotope labeling was to explore the mechanism by which the fatty acyl composition of membrane phospholipids was altered quickly after a downward temperature-shift in Tetrahymena pyriformis. 1. When 39 degrees C-grown Tetrahymena cells were shifted to 15 degrees C, unsaturated fatty acids, especially gamma-linolenic acid, were increased, with compensating decrease of palmitic acid in phospholipids. 2. However, in the [32P]Pi-prelabeled cells, the specific radioactivities of major membrane phospholipids, phosphatidylethanolamine, phosphatidylcholine and 2-aminoethylphosphonolipid, were not changed within 10 h after shift-down. Furthermore, in the [14C]palmitic acid-prelabeled cells, the specific radioactivities toward phospholipids also did not change after temperature-shift. 3. After the shift, the rate of incorporation of [14C]acetate into fatty acids was reduced to less than one-tenth and the principal fatty acid newly synthesized was linoleic acid, and palmitic and gamma-linolenic acids were also formed to almost same degree. 4. In contrast, when cells prelabeled with both [32P]Pi and [14C]palmitic acid were shifted to 15 degrees C and then linoleic and gamma-linolenic acids, which are known to increase during cold acclimation, were added to the medium, the 14C/32P ratios of major phospholipids were progressively decreased until 5 h after the shift. These results suggest the mechanism for the alteration of phospholipid fatty acyl composition at lowered growth temperature by which preexisting fatty acids of membrane phospholipids would be deacylated and then, after modification to adequate unsaturated fatty acids by desaturation and/or elongation, be reacylated again into lysophospholipids.

Mechanism for adaptive modification during cold acclimation of phospholipid acyl chain composition in Tetrahymena. II. Activities of 2-acyl-sn-glycerol-3-phosphorylcholine and 2-acyl-sn-glycerol-3- phosphorylethanolamine acyltransferases involving the reacylation.

The deacylation-reacylation process is very important for the alteration of phospholipid fatty acyl composition on lowering of growth temperature in Tetrahymena pyriformis (Kameyama, Y., Yoshioka, S. and Nozawa, Y., (1984) Biochim. Biophys. Acta 793, 28-33). Microsomes isolated from Tetrahymena cells have reacylation activities not only for 1-acyl-sn-glycerol-3-phosphorylcholine (1-acyl-GPC) and 1-acyl-sn-glycerol-3-phosphorylethanolamine (1-acyl-GPE) but also for 2-acyl-GPC and 2-acyl-GPE. Unsaturated fatty acyl-CoAs were in general much better substrates than saturated fatty acyl-CoAs for acylations of 1-acyl-GPC and 1-acyl-GPE. The acylation rates for 1-acyl-GPE were almost the same in palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA and gamma-linoleoyl-CoA. However, the acylation activity for 1-acyl-GPC was more than 2-fold higher with palmitoleoyl-CoA than with any other unsaturated fatty acyl-CoAs. In contrast, both 2-acyl-GPC and 2-acyl-GPE acyltransferases did not show a distinct preference for various acyl-CoAs, although palmitoyl-CoA was incorporated into both 2-acylphospholipids at higher rates than into 1-acylphospholipids. These specificities for various acyl-CoAs of 1-acyl- and 2-acyl-GPC and 1-acyl- and 2-acyl-GPE acyltransferases were not changed in the microsomes isolated from cells grown isothermally at 39 degrees C and 15 degrees C and cells shifted from 39 degrees C to 15 degrees C. However, the acylating ratio of linoleoyl-CoA to palmitoyl-CoA, which were chosen as typical unsaturated and saturated fatty acyl-CoAs, in the microsomes from cells grown at 15 degrees C was 1.5-3.0-times higher than in the microsomes from 39 degrees C-grown cells in four acyltransferase activities. These results suggest that the changes of acyl-CoA specificities in reacylation enzyme activities during temperature down-shift would make little contribution to the increase in unsaturated fatty acids in phospholipids, although reacylating enzymes from isothermally grown cells at lower temperature would partially participate in those increases.

The occurrence of direct desaturation of phospholipid acyl chain in Tetrahymena pyriformis. Thermal adaptation of membrane phospholipid.

Microsomes from Tetrahymena pyriformis catalyzed the conversion of 1-acyl-2-[1-14C]oleoyl-sn-glycero-3-phosphorylcholine to 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphorylcholine in the presence of oxygen and NADH or NADPH as cofactors. This desaturation enzyme activity was inhibited by cyanide and increased by 0.05-0.1% Triton X-100. Under optimal conditions desaturation appeared to follow Michaelis-Menten kinetics with a Km value of 6.9 . 10(-4) M. During incubation, no significant cleavage of phospholipid substrate was observed and no desaturation of free fatty acid occurred. The activity of 1-acyl-2-oleoyl-sn-glycero-3-phosphorylcholine desaturase was increased approx. 4-fold when Tetrahymena cells were shifted to a lower growth temperature. These data suggest the existence of a direct phospholipid desaturation system from oleoylphosphatidylcholine to linoleoylphosphatidylcholine. In addition, this desaturation may participate in the control of membrane lipid adaptation to a lower growth temperature in Tetrahymena.

Ca(2+)-independent phospholipase A2 activity associated with secretory granular membranes in rat parotid gland.

Phospholipase A2 activity was detected in a secretory granular fraction (SG) purified by Percoll gradient centrifugation from rat parotid gland using [3H]phosphatidylcholine (PC) as a substrate. High activity of this enzyme was observed at neutral pH. The enzyme was activated by Triton X-100 and did not require Ca2+ for its activity. In the absence of Ca2+, its apparent Km for exogenous PC was 28 microM while it was slightly increased by adding 5 mM CaCl2 (73 microM). Furthermore, the enzyme was located essentially in a granular membrane fraction separated from granular lysate. The deacylation activities were also detected in other subcellular fractions, which showed a different detergent-susceptibility or pH-dependency from that in SG. These results suggest that secretory granules have membrane-bound phospholipase A2 which has properties different from that found in other organelles.

Possible involvement of 1-acyl-glycerophosphorylinositol acyltransferase in arachidonate enrichment of phosphatidylinositol in human platelets.

Microsomes isolated from human platelets synthesize phosphatidylinositol by the action of acyl-CoA: 1-acyl-sn-glycerol-3-phosphorylinositol(1-acyl-GPI) acyltransferase. The properties of 1-acyl-GPI acyltransferase were compared with those of 1-acyl-glycerophosphorylcholine (1-acyl-GPC) acyltransferase. Apparent Km values of 1-acyl-GPI and 1-acyl-GPC acyltransferases for the corresponding acyl acceptor (lysophospholipid) were 22 and 20 microM, respectively, in the presence of arachidonoyl-CoA as fatty acyl donor. However, the Km value (1.3 microM) of 1-acyl-GPI acyltransferase for arachidonoyl-CoA was much lower than that (5.0 microM) of 1-acyl-GPC acyltransferase. Under optimal conditions, the acylation rate of 1-acyl-GPI with arachidonoyl-CoA was 2-6 times higher than with oleoyl-CoA and linoleoyl-CoA, and was very low with saturated fatty acyl-CoAs. The acylation rates with various acyl-CoAs for 1-acyl-GPI were different from those for 1-acyl-GPC. These results suggest that the reacylation pathway of 1-acyl-GPI participates in the incorporation of arachidonic acid to phosphatidylinositol in platelet microsomes. Furthermore, there were no significant effects of thrombin-activation on acyl-CoA specificity for 1-acyl-GPI and 1-acyl-GPC acyltransferase in human platelets.

Studies of diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities in Tetrahymena microsomes.

Microsomes isolated from Tetrahymena pyriformis synthesized phosphatidylcholine and phosphatidylethanolamine by CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1), utilizing ethanol-dispersed dioleoglycerol. Cholinephosphotransferase and ethanolaminephosphotransferase activities have similar dependences on MgCl2 and MnCl2, but the latter was more effective than the former for both enzyme activities. The V values for 1,2-dioleoylglycerol obtained at optimal conditions were 1.8 nmol/min per mg microsomal protein for cholinephosphotransferase and 0.6 nmol/min per mg microsomal protein for ethanolaminephosphotransferase. Both enzymes could not utilize 1,3-dioleoylglycerol or 1-oleoylglycerol as substrates. Cholinephosphotransferase had an apparent Km for CDPcholine of 11.7 microM with 1,2-dioleoylglycerol and was inhibited by CDPethanolamine competitively. On the other hand, ethanolaminephosphotransferase has an apparent Km for CDPethanolamine of 8 microM and CDPcholine was a noncompetitive inhibitor of ethanolaminephosphotransferase activity. Furthermore, despite the marked alteration of phospholipid composition occurring during the temperature acclimation of Tetrahymena cells, both enzyme activities showed similar dependences on growth and incubation temperatures. This may imply that the final step of de novo synthesis of two major phospholipids does not participate in the thermally induced modification of the profile of phospholipid polar head group in membranes.

Age-dependent modifications in membrane lipids: lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes.

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.

Phospholipid metabolism in rat submandibular gland. Positional distribution of fatty acids in phosphatidylcholine and microsomal lysophospholipid acyltransferase systems concerning proliferation.

Rat submandibular gland phosphatidylcholine mainly consisted of the 1-saturated acyl-2-unsaturated acyl type. The high occupancy of unsaturated fatty acid at the C-2 position is in part explained by the preference of microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) acyltransferase for unsaturated fatty acyl-CoAs. This enzyme activity was partially inhibited by divalent cations. Ca2+ may be important for regulation of a deacylation-reacylation cycle, suggested because Ca2+ is also known to activate the deacylation enzyme, phospholipase A2. Although the presence of 1-acyl-GPC acyltransferase activity is also observed in plasma membrane of the submandibular gland, the microsomal enzyme showed properties different from the enzyme in plasma membrane in terms of its susceptibility to neural salts and detergents. Cell proliferation caused by chronic administration of isoproterenol resulted in an increase of linoleic acid at the C-2 position of phosphatidylcholine. However, this alteration did not correlate with the changes of activity and substrate specificity of 1-acyl-GPC acyltransferase and the other C-2 acylation enzyme, 1-acyl-sn-glycero-3-phosphate acyltransferase, which suggests that the alteration of fatty acid by isoproterenol treatment is due to a change of supply of substrates or specific acyl breakdown of phosphatidylcholine.

Substrate specificity of microsomal 1-acyl-sn-glycero-3-phosphoinositol acyltransferase in rat submandibular gland for polyunsaturated long-chain acyl-CoAs.

Microsomal 1-acyl-sn-glycero-3-phosphoinositol (1-acyl-GPI) acyltransferase in the rat submandibular gland showed the highest specific activities for eicosanoid-related polyunsaturated acyl-CoAs, such as arachidonoyl-, bishomo-gamma-linolenoyl- and 5,8,11,14,17-eicosapentaenoyl-CoAs, with low Km values. High activities were also obtained with acyl-CoAs having long (more than 14 carbon atoms) and n - 6 unsaturated (more than 3 double bonds) acyl chains. This enzyme also utilized acyl-CoAs having trans-unsaturated or branched chains, but not short-chains, as substrates, although the activity levels for trans-unsaturated acyl-CoAs were lower than those for cis-unsaturated acyl-CoAs. Chronic administration of isoproterenol induced decreases of this enzyme activity and the content of arachidonic, bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids at the sn-2 position of phosphatidylinositol. These results suggest that enrichment of arachidonic acid in the sn-2 position of phosphatidylinositol is established by the high specificity and affinity of 1-acyl-GPI acyltransferase for arachidonoyl-CoA. On the other hand, the low level of bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids in the sn-2 position of phosphatidylinositol may be explained by their limited availability.

Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP.

A secretory granular fraction (SG) and a plasma membrane rich fraction (PM) have been isolated from rat parotid gland by differential and Percoll gradient centrifugation. With these two fractions, a cell-free interaction system has been reconstituted to clarify the exocytotic interaction between the secretory granules and plasma membranes, and the conditions of amylase release from SG have been characterized in vitro. The addition of PM into this assay system induced a rapid and transient release of amylase from SG. Some other membranes such as erythrocyte ghosts also mimicked the effect of PM. This release was increased by Ca2+, but was not completely blocked by EGTA. Simultaneous addition of 1 mM ATP with 1 mM MgCl2 (Mg-ATP) in the presence of Ca2+ reduced this release. However, in spite of the existence of Mg-ATP, the stimulation of PM-induced amylase release was caused by Ca2+ in a concentration-dependent manner (10(-7)-10(-3) M). These results suggest that Ca2+ and Mg-ATP should participate as important regulators in the exocytotic interaction between secretory granules and plasma membranes in this system. Furthermore, the differences between our system and intact cells are also discussed.

Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading.

The influence of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the bone response to mechanical loading was evaluated. Six-month-old female Wistar rats were used and assigned to three groups (n = 12/group): Vehicle administration (EP4-V), low-dose ONO-4819 administration (EP4-L, 3 microg/kg BW), and high-dose ONO-4819 administration (EP4-H, 30 microg/kg BW). ONO-4819 was subcutaneously injected in the back twice a day for 3 weeks. Loads on the right tibia at 39.4 N for 36 cycles at 2 Hz were applied in vivo by 4-point bending every other day for 3 weeks. Whole-body bone mineral content showed a significant difference between EP4-V and EP4-H (P < 0.05). Bone mineral density (BMD) of the total and regional tibia (the region with maximal bending at the central diaphysis) was higher in EP4-H than EP4-V, showing a significant effect of loading (P < 0.001) and ONO-4819 (P < 0.05). BMD of the total femur was higher in EP4-H than EP4-V (P < 0.01) and that of the distal femur was higher in EP4-H than EP4-V (P < 0.001). Histomorphometry of the cortical bone showed that loading increased formation surface (FS/BS), mineral appositional rate (MAR), and bone formation rate (BFR/BS) significantly at the lateral periosteal surface (P < 0.001); however, the effect of ONO-4819 was not significant. At the medial periosteal surface, loading increased the three parameters (P < 0.001) and ONO-4819 increased FS/BS (P < 0.001) and MAR (P < 0.05) significantly. At the endocortical surface, the effects of both loading and ONO-4819 were significant on all three parameters (for loading; FS/BS P < 0.01, MAR P < 0.05, BFR/BS P < 0.03, for ONO-4819 all P < 0.001). It was concluded that ONO-4819 increased cortical bone formation in rats and there was an additive effect on the bone response to external loading by 4-point bending.

DNA vaccine against hamster oral papillomavirus-associated oral cancer.

Previously we developed a carcinogenesis model involving the combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Enhanced anti-tumour effect of cisplatin with low-voltage electrochemotherapy in hamster oral fibrosarcoma.

The aim of this study was to determine the effects of low-voltage electrochemotherapy with intraperitoneal cisplatin on hamster oral fibrosarcoma. Oral fibrosarcoma was transplanted submucosally into the cheek pouch mucosa of 100 hamsters. After transplantation, the hamsters were randomly divided into four equal groups. These groups received no treatment (D-E-); 2 mg/kg body weight cisplatin treatment without electroporation (D+E-); electroporation without cisplatin treatment (D-E+); or 2 mg/kg body weight cisplatin treatment followed by electroporation (D+E+). Electrical pulse treatment together with cisplatin injection markedly reduced the size of the tumour, whereas cisplatin injection or electrical pulse treatment alone did not. These results clearly indicate that the anti-tumour effect of cisplatin on hamster oral fibrosarcoma was considerably potentiated or enhanced by the administration of local electrical pulses at low voltages.

Microsomal diacylglycerol acyltransferase in rat parotid and submandibular glands: acylation of 1,2-dioleoyl-sn-glycerol dispersed with phospholipids.

In order to investigate microsomal diacylglycerol acyltransferase activity, ethanol or several detergents have been used as a dispersing agent for water-insoluble substrates. However, ethanol acyltransferase interferes with the activity of this enzyme, and detergents inhibit it. We examined the properties of microsomal diacylglycerol acyltransferase in rat salivary glands without detergents or organic solvents. 1,2-Dioleoyl-sn-glycerol (1,2-diolein) was dispersed by sonication. The activity was measured as the formation rate of [14C]triglyceride using [1-14C]palmitoyl-CoA as an acyl-donor. The reaction was dependent on the microsomal protein and 1,2-diolein at least up to 145 micrograms/ml and 3.6 mM, respectively. The specific activities were 3.91 +/- 0.57 and 3.80 +/- 0.77 nmol/min per mg protein (SEM, n = 4) in the parotid and submandibular glands, respectively. They were 12- to 20-fold higher than the activities in liver, brain and spleen, and two orders of magnitude higher than that assayed with microsomal endogenous diacylglycerol. Adding tissue phospholipids to 1,2-diolein suspension reduced the concentration of 1,2-diolein required for the maximal velocity. A similar, but reduced, effect was induced by egg yolk phosphatidylcholine in place of the tissue phospholipids. The level of activity was recovered by adding another phospholipid class to the phosphatidylcholine. The results suggested that the physical condition of the substrate diacylglycerol affects diacylglycerol acyltransferase activity in rat salivary gland microsomes.

Epithelial cell proliferation in oral lichen planus.

Although the pathogenesis of oral lichen planus (OLP) is not clear, a small proportion of cases with OLP are reported to transform to cancer. We examined the epithelial cell proliferation status of OLP to relate the labelling index to microscopic features surveyed routinely in pathology. Mucosal biopsies obtained from 44 cases diagnosed with OLP with an intact oral epithelium and 10 normal control specimens from Japanese subjects were immunohistochemically stained with MIB and p53 antibodies. The Ki67 labelling index (LI) was significantly higher in OLP compared with normal controls. A particularly large number of OLP lesions (64%) were p53 positive. No association was, however, found with p53 expression and the Ki67 LI. Atrophic and flat epithelia had a quantitatively higher LI, which did not significantly differ from acanthotic biopsies. Increased cell proliferation in OLP is likely to be a secondary phenomenon due to the damage inflicted on keratinocytes by infiltrating mononuclear cells in the submucosa.